Topical viral infections are very common in human beings as the first viral entry into the body is often through virus contact with the external body surface.
A virus is a very small infectious agent with varying diameter between 10 to 300 nanometers and varying length up to 1400 nm (filoviruses) or more. The viruses can reproduce only inside the host cell.
All viruses have genes made from either DNA or RNA, and a protein coat that protects these genes. Some viruses have an envelope of fat that surrounds them when they are outside the host cell.
Although the mechanism of viral entry into the host cell varies among different virus groups, all viruses follow three basic steps to infect the host cell and to reproduce. Once the virus comes in contact with a host cell, the first step is the viral attachment to the host cell (initial infection), the second is the replication of viral genome inside the cell (virus multiplication) and the third is the exit of newly generated virus particles from the host cell.
The mode of progression of a topical viral infection is completely different than a systemic viral infection.
During a topical external infection such as the influenza virus, initially a few virus particles come in contact with a few cells on the skin or the mucus membrane and enters the cells. In some cases, the virus may be present in the body as a latent virus which migrates towards the skin or the mucus membrane. For example, in case of labial or genital herpes virus infection, the latent virus present in the nerve cells migrates towards the labial skin or towards the vaginal mucosa and start multiplying in a few cells. In other cases, once entered, some viruses can remain dormant into the cells (such as Herpes I and II into the nerve cells), and create the disease once the body defense mechanisms are dull. In all cases, there are practically no clinical signs at this stage.
After initial infection, the virus multiplies in a few cells and millions of new virus particles are then liberated topically and they start attacking new cells to create visible lesion. The same process occurs for the throat viral infections with the exception that the initial virus particles are not stored in the body but are inhaled by the host person.
The virus envelope has an important role for virus initial infection.
The virus envelope is associated with several proteins (glycoproteins) of the surface coat.
Glycoproteins of the surface coat are transmembrane proteins, anchored to the envelope by a hydrophobic domain. They are visible on the virus surface. Virus uses these glycoproteins to attach to the cell membrane and to enter into the host cell.
The virus envelope is a highly complex structure containing many types of glycoproteins. For example the herpes simplex virus (HSV 1 and 2) contains surface glycoproteins such as gB, gD, gH, gL, gC, gI, gE as main proteins. To infect the cells, currently it is postulated that the C and/or B glycoproteins (gC and gB respectively) and probably the gH glycoprotein, bound to the heparan sulfate receptors on the cell surface, to fuse with the host cell membrane and to create an opening or pore, through which the virus enters the host cell. Therefore, cells that are devoid of heparan sulfate receptors are not susceptible to HSV (Natalia Chesenko, 2002). This specificity determines the host range of a virus.
The differences in the surface glycoprotein structures confer different morphology and antigenicity in the same family of virus. For example, three different types of influenza virus, dubbed A, B, and C have been identified with HA (hemagglutinin) and NA (neuraminidase) as main surface glycoproteins. 13 major types of HA and 9 major antigenic determinants of NA have already been identified. This shows that the viral capsid may contain a very large variety of glycoproteins on the surface coat. It also means that each type of virus has its own way to facilitate cell attachment and cellular entry.
All the viral glycoproteins are not yet discovered and there is continuous research on the presence of new virus glycoproteins and their role in viral multiplication and infection. Continuous discovery of new virus surface glycoproteins, their role in host cell infection and frequent viral mutations makes the development of a specific topical antiviral drug nearly impossible.
In the absence of any specific antiviral drug, vaccines are currently considered the best antiviral therapy.
Vaccines may contain virus glycoprotein subunits but they have poor immunity as all the glycoproteins are not represented in a vaccine and their antigenicity is variable.
The whole inactivated virus vaccines are more efficient but can be prepared only for small and highly antigenic viruses.
Live virus vaccines with reduced viral pathogenicity are good immunogens but are unstable, dangerous (live viruses) with a risk of reversion to virulence.
Unfortunately due to complex surface glycoprotein structures, vaccines cannot be developed for many viruses such as the, HIV, herpes, papilloma virus and many others.
Most of the antiviral treatments are directed to interfere with intracellular virus multiplication process but have no effect at all on free virus particles, when the virus particles are present on the skin lesions (labial herpes for example), vaginal mucosa (genital herpes for example), or throat surface (influenza for example).
All the currently available antiviral drugs are directed to stop intracellular virus growth once the infection is already established. For example, intracellular nuraminidase inhibitors are used to treat the influenza, A, B and C type of enveloped viruses with 2 main classes of drugs by oral route:                the adamantanes which interfere with viral uncoating inside the cells and are effective only against influenza A type of viruses;        the newer class Zanamivir or Oseltamivir (Tamiflu) which interfere with the release of intracellular progeny viruses and require early oral administration to stop further virus growth.        
To treat systemic herpes virus infection, different types of Acycloguanosines (Aciclovirs®) are used and are marketed under trade names such as Zovirax®, Ciclovir®, Herpex®, Acivir®, Acivirax®, Aciclovir® and Zovir®. These are nucleoside analogues which interfere with virus growth inside the cells but have no effect on the free virus on the surface of the body as these drugs are transmitted through the circulation and cannot reach the free virus particles present on the open viral lesions.
Furthermore, as the entire antiviral drugs act by interfering with normal cellular metabolic functions, they often induce severe toxic effects. Oral administration of Acyclovirus® and Famciclovir® for the treatment of labial herpes, genital herpes, herpes zoster and chickenpox may induce nausea, vomiting, diarrhea, headache, rashes, kidney damage and confusion; Amantadine® for the treatment of influenza A may cause nausea, loss of appetite, nervousness, light headache, unsteadiness, sleepliness, and confusion; Cidofovir®, Ganciclovir® and Foscarnet® for the treatment of cytomegalovirus may produce kidney damage and low white blood cell count; interferon-alpha given to stimulate immunity against viral infections may cause flu like symptoms, anaemia, depression, low white blood cell count, low platelet count; Oseltamivir® for the treatment of influenza A & B is known to produce nausea, vomiting and dizziness, and Ribavirin® used for RSV infection in children may produce breakdown of red blood cells and anaemia (URBAN M, Merck Manual, 2009). Topically applied acyclovirus (Zovirax®, Penciclovir® Vidarabine®) for cold sores are not effective and have no severe toxic effects known.
Neutralizing the free virus particles topically on the body surface seems to be a very efficient alternative to stop the infection; however there is currently no vaccine available to stop topical viral infection, in particular at the stage of the initial infection, to avoid topical viral entry into the cells.
Recent scientific work also proves that in addition to the virus surface glycoproteins, proteases also play an important role in facilitating topical viral entry into the cell. The use of proteases during viral multiplication inside the cells was known but their involvement to facilitate the viral entry from external body surface, such as the herpes and the influenza virus entry from the skin lesions or from the throat surface, was discovered recently (Kido, 2007; Delboy, 2008).)
As protease have the potential to hydrolyze specific proteins, some viruses having no specific receptor on the surface to attach and to enter into the host cell, take the help of these proteases to break the cell wall and to enter into the cells.
Proteases, also known as proteinases or proteolytic enzymes, are a large group of enzymes found in or outside the cells, particularly in the vicinity of the damaged tissues and play a vital role in protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain.
Proteases are involved in the splitting of the protein molecules. Their main role is to break and to clean the proteinous debris generated during the tissue breakdown because such substances interfere with the tissue repairing process. They are essential to create a favorable environment for subsequent tissue repairs. Some of them can detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidases) while the others attack internal peptide bonds of a protein (endopeptidases such as trypsin, chemotrypsin, pepsin, papain, elastase).
There are hundreds of proteases found topically or inside the cells and can be divided into four major groups according to the character of their catalytic active site and conditions of action: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases or Matrix-Metallo-Proteins (MMPs). Attachment of a protease to a certain group depends on the structure of catalytic site and the amino acid (as one of the constituents), essential for its activity.
Many proteases may be found topically on a virus infected skin or mucus membrane such as pepsin, trypsin, chymotrypsin, subtilisin, cystin proteinase (cathepsin B,H,K,L,S), aspartic proteinase (cathepsin D), clotting factors, and matrix metalloproteins involved in the process of topical wound healing such as gelatinase A (MMP-2), gelatinase B (MMP-9), collagenase (MMP-1), collagenase 3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-3 (MMP-11), MT-1 MMP (MMP-14), macrophase metalloelastase (MMP-12) and surely many others which are not yet discovered.
The analyses of different type of proteases present in the extra cellular matrix, in the virus lesions on the skin (example herpes labialis), on the mucus membrane (example genital herpes), and on the throat surface (example influenza virus) also indicate that there are many different type of proteases present at the site of tissue infection having specific proteolytic activity.
It is well established that many viruses use proteases to facilitate their multiplication inside the cells such as the HIV 1 protease which helps HIV-AIDS virus to multiply by cleaving newly synthesized polyproteins at the appropriate places to create the mature protein components of an infectious HIV virion. Without effective HIV protease, the HIV virion remains non-infectious.
Therefore, intracellular protease inhibitors such as Saquinavir®, Ritonavir®, Indinavir®, Nelfinavir® and several others were developed to block the HIV virus growth. These products are given orally, either alone or in combinations (example Liponivir® and Ritonavir®) to block HIV virus growth. But their use as a general antiviral is highly limited due to their toxicity, side effects and poor activity.
Some intracellular protease inhibitors such as Agenerase®, Crixivan®, Invirase®, Kaletra®, Lexiva®, Norvir®, Prezista®, Reyataz® and Vitacept® have already been authorized as anti-protease drugs and are used orally in combination with other antiviral drugs for the treatment of AIDS.
Unfortunately most of these protease inhibitors are proteins in nature and are easily destroyed by the natural proteases present in the wound (Patick et al. 1998) and therefore they cannot be used for topical viral infections.
All the future antiviral treatments are also directed to stop intracellular virus replication by oral administration such as new protease inhibitors, nucleoside RNA replicase inhibitors, integrase inhibitors, nucleoside reverse transciptase inhibitors, non-nucleoside reverse transcriptase inhibitors and cycloporin derivatives but due to their intracellular mode of action, their topical use to stop virus entry into the cell is not yet envisaged.
Due to their role in tissue repair, topical concentration of proteases increases markedly at the site of injury. As topical viral infections are usually not detected before the skin or the mucosa is damaged, the amount of proteases is very high in all the topical viral infections.
The influenza virus which infects more than 500 million people in the world every year, which initially comes in contact with the throat cells during inhalation, has no processing protease for the viral membrane fusion glycoprotein precursor and the virus cannot enter into the cells. This virus therefore uses proteases present on the surface of the respiratory tract to enter and to infect throat cells. At least 5 different processing proteases (trypsin-like) have already been identified in the airways (Kido, 2007). Initially, a few virus particles enter into the cells, multiplies and millions of new virus particles are then liberated onto the throat surface. All these virus particles need the help of proteases to enter into new healthy cells and to spread the infection. Therefore our body defense mechanisms liberate anti-proteases called secretory leukoproteases in the upper respiratory tract and the pulmonary surfactants in the lower respiratory tract to reduce the amount of free proteases available for viral entry. When proteases activity predominant over the activities of inhibitory compounds, virus infection cannot be stopped (Kido, 2004). Recent scientific research also indicate that other topical viruses such as the herpes virus which cause labial and genital herpes also take the help of some proteases to infect the cells.
It has been shown that the open herpes virus lesions, the level of MMP-2 and MMP-9 proteases increase during early infection (Martinez, 2004), that certain protease inhibitors reduce the activity of herpes virus (La Frazia, 2006), and that cellular proteases are involved in the pathogenicity of enveloped viruses (Kido 1996).
Taking into consideration the role of surface viral glycoproteins for viral attachment with the host cell and the use of proteases by the viruses to enter into the host cells, it is essential to find a method for treating topical viral infections by neutralizing virus surface glycoproteins and the corresponding proteases simultaneously.
Currently there is no known topical anti-protease inhibitor or virus surface glycoprotein inhibitor to block topical virus entry into the cells.
This may be related to the fact that as there are many proteases, each with a specific activity, it is extremely difficult to find a specific drug which can neutralize all the proteases.
In the absence of current knowledge regarding exact number of proteases present topically and their role in topical viral infection, only a non-specific protease inhibitor can block all the proteases.
Taking into consideration the amount of free virus particles present topically on an infected surface (in millions), an effective treatment should not only neutralize specific virus glycoproteins but should also minimize the amount of those proteases which facilitate viral entry into the cells.
In the absence of any specific drug to kill topical virus particles which continue infecting new cells, Acyclovir® containing creams are also now marketed for topical application but due to their intracellular mode of action Acyclovir® cannot act on free virus particles liberated topically in the lesion and which are present outside the cell. In all the topical viral infections, the free virus particles present on the surface of the lesion continue attacking new healthy cells from outside to spread the infection.
Some herbal preparations are also proposed to apply topically on viral lesions but the absence of widespread use of any such herbal drug itself proves that their efficiency is extremely limited and they cannot be considered as specific antiviral drugs.
For example, the use of Rhubarb with Sage plant extract was proposed for the treatment of herpes labials where clinical results showed the mean lesion healing time of 7.6 days with sage cream, 6.7 days with Rhubarb-sage cream and 6.5 days with Zovirax® (Saller et al 2001). The association of these two plant extracts reduced the herpes lesion healing time by approximately 12% compared to about 15% with Acyclovir® containing Zovirax® cream which is not at all satisfactory to consider the product as a specific topical herpes treatment.
A large number of plant extracts have also been tested using isolated in vitro cell culture models or on the specific virus attachment receptors but they were not considered sufficiently antiviral for further clinical trials or for further development as antiviral drugs.
Sulfated polysaccharides from sea algae were shown to interfere with HIV heparin receptor binding (Witvrouw, 1997); Epigallocatechin gallate from green tea was found to block gp120-cd4 binding of the HIV-1 virus in isolated cells (Hamza, 2006); the extracts of Chinese medicinal plants Prunella vulgaris and Rhizoma cibotte were shown to inhibit HIV-1 gp41 Six-helix bundle formation in vitro probably as a result of tannin interference with HIV-1 gp41 Six-Helix bundle formation (Liu, 2002); the extract of an Indian plant Swertia chirata was found to reduce HIV-1 grown in vitro indicating that some plant extracts were found to have some anti-viral activity but this topical antiviral effect is not enough to use the plant as an anti-viral drug.
Other natural substances such as propolis was also suggested to inhibit herpes simplex virus type II growth as the aqueous extract rich in phenyl carboxylic acid and poor in flavonoids and the ethanolic extract rich in flavonoids, both showed reduction in HSV-2 infectivity (Nolkemper, 2009).
As in all the topical viral infections the superficial skin or mucus cells initially liberate a large amount of free virus particles on the surface of the lesions and as this free virus attach new healthy cells, often with the help of various proteolytic enzymes, an ideal topical antiviral treatment should possess the following main properties:                It should neutralize all the viral glycoprotein receptors which help the virus particles to enter into the cell so as to stop new cell infection.        It should simultaneously neutralize all of the proteases present on the infected surface which may be used by the viruses to enter into the cell membrane.        It should be non-irritant, non toxic to the skin or mucus cells and should be inexpensive.        